Pluripotent stem cells offer great promise for regenerative medicine, but it remains a significant challenge to finely control their activities to build complex organs de novo. Tissue engineering has approached this problem by designing synthetic scaffolds to control stem cell function, but production of true tissue mimics in this fashion is a daunting task. Animals that have evolved mechanisms of adult tissue regeneration provide an opportunity to discover how stem cells can be naturally instructed to undergo post-embryonic organogenesis. Planarian flatworms are famous for their ability to regenerate any missing tissue by controlling the activity of pluripotent stem cells termed neoblasts. Because such animals can engage a multitude of different regenerative programs dependent on the nature and extent of injury, they require exquisite control over the utilization of stem cells. Thi ability likely either resides in novel signaling pathways or in a unique use for well-described signaling pathways. This proposal describes a strategy for specific identification of the regulatory molecules that control regenerative growth by stem cells in planarians. The analysis of the function of these genes using RNA interference will describe pathways that control regenerative growth. Ultimately, a quantitative understanding of stem cell control in regeneration will be necessary to efficiently adapt natural regenerative mechanisms to the enhancement of human tissue repair. The proposal further describes the application of single-molecule fluorescence in situ hybridization to quantitatively analyze the spatial and temporal dynamics of Wnt, BMP and hedgehog signaling in planarian regeneration. This approach will allow a systems-level identification of signal control mechanisms that underlie stem cell-mediated organogenesis through regeneration. It is likely that stem cells and tissue repair mechanisms are ancient. Therefore, these studies have the potential to identify novel conserved proteins that could be modulated to enhance tissue repair and uncover basic principles of tissue restoration that could be applied to regenerative medicine.